Protocol for identification and validation of 2'3'-cGAMP-binding proteins by photoaffinity probes

Yingjie Hou; Heng Lu; Le Sun; Yaoyang Zhang; Hong Jiang

doi:10.1016/j.xpro.2021.101076

Protocol for identification and validation of 2'3'-cGAMP-binding proteins by photoaffinity probes

STAR Protoc. 2022 Jan 10;3(1):101076. doi: 10.1016/j.xpro.2021.101076. eCollection 2022 Mar 18.

Authors

Yingjie Hou^{1

2}, Heng Lu^{1

2}, Le Sun^{1

2}, Yaoyang Zhang^{1

2}, Hong Jiang^{1

2}

Affiliations

¹ Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China.
² University of Chinese Academy of Sciences, Beijing 100049, China.

Abstract

Mammalian cyclic dinucleotide 2'3'-cGAMP functions as a second messenger in innate immune response. Here, we report a protocol to utilize 2'3'-cGAMP photoaffinity probes to capture 2'3'-cGAMP-binding or 2'3'-cGAMP-interacting proteins from HeLa cell lysate for in-gel visualization by fluorescent imaging or identification by SILAC-based quantitative MS. Further validation is also executed using photoaffinity probes to demonstrate the direct interaction of 2'3'-cGAMP with purified target proteins in vitro or endogenous target proteins in 293T cells. For complete details on the use and execution of this profile, please refer to Hou et al. (2021).

Keywords: Cell Biology; Chemistry; Immunology; Mass Spectrometry; Molecular/Chemical Probes; Protein Biochemistry.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Carrier Proteins* / metabolism
HeLa Cells
Humans
Mammals / metabolism
Nucleotides, Cyclic*
Second Messenger Systems

Substances

Carrier Proteins
Nucleotides, Cyclic
cyclic guanosine monophosphate-adenosine monophosphate